Method for extracting soluble proteins from microalgal biomass

ABSTRACT

A method for preparing a protein isolate of the biomass of microalgae of the genus  Chlorella , includes the following steps: supplying a microalgal biomass produced by fermentation, washing the biomass so as to eliminate the soluble interstitial compounds and concentrating the biomass, mechanically grinding the washed and concentrated biomass in a horizontal ball grinder-type system in order to produce an emulsion, destructuring the emulsion thus produced, triple-phase separation so as to separate the soluble fraction from the fractions containing the lipids and the cell debris, recovery of the soluble fraction thus produced in order to produce the soluble protein isolate, then evaporation, pasteurization and atomization of the protein isolate.

The present invention relates to a method for extracting soluble proteins from microalgal biomass.

The present invention also relates to the microalgal protein isolates obtained in this way.

PRESENTATION OF THE PRIOR ART

It is well known to those skilled in the art that Chlorellae are a potential source of food, since they are rich in proteins and other essential nutrients.

They are described as containing 45% of proteins, 20% of fats, 20% of carbohydrates, 5% of fibers and 10% of minerals and vitamins.

Given their abundance and their amino acid profile, microalgal proteins are thus considered as an alternative source to soy or pea proteins in food.

The protein fraction may also be exploited as a functional agent in the cosmetic, or even pharmaceutical, industries.

However, developments in food applications for microalgal proteins have not been significant, since the presence in said fractions of undesirable compounds (such as chlorophyll) leads to undesired changes in color, flavor and structure of the food compositions containing them.

To increase their potential in food applications and also to increase their commercial value, these proteins must be extracted from the microalgae without affecting their molecular structure.

“Soft” extraction techniques would therefore be necessary to isolate proteins with high solubilities and good technical and functional properties, but the rigidity of microalgal cell walls, especially of green microalgae, is fundamentally in contradiction to this, since it disrupts the extraction and integrity of the intracellular proteins.

Thus, on the contrary, conventionally “hard” physical or chemical conditions are employed to break the microalgal cell wall.

Numerous studies thus propose technologies of extraction by organic solvent type or high-pressure homogenization type.

In these technological choices, the denaturing of proteins was not however considered to be bothersome, since most of these methods were developed for purposes of analyses or intended to provide a substrate for the enzymatic digestion producing protein hydrolyzates.

However, an effective disintegration method preserving the integrity of the cell components should maximize not only the yield, but also the quality of the products extracted.

In other words, a method for optimized disintegration of the wall must for example avoid:

-   -   chemical contamination of the targeted products,     -   using a breaking energy which is too high; the latter possibly         causing irreversible denaturation or degradation of the         intracellular molecules of interest.

Moreover, for large-scale productions, it is important for the process chosen to be transposable to this scale.

Finally, the introduction of this cell disintegration step must be easy and must not have a negative impact on the subsequent method/treatment steps.

All these limitations influence the efficiency of the disintegration method and by the same token its energy consumption.

This is why the bead mill technology is preferred, since it is considered to be efficient for releasing intracellular proteins in their native form.

In a bead mill, the cells are agitated in suspension with small spherical particles. The breaking of the cells is caused by the shear forces, the milling between the beads, and the collisions with beads.

The description of an appropriate bead mill is, for example, given in the U.S. Pat. No. 5,330,913. These beads break the cells so as to release the cell content therefrom. A suspension of particles of smaller size than the cells of origin is then obtained in the form of an “oil-in-water” emulsion.

This emulsion is generally atomized and the water is eliminated, leaving a dry powder containing, however, a heterogeneous mixture composed of cell debris, interstitial soluble compounds, and oil.

The difficulty to be solved in the use of these cell disintegration technologies is the isolation of solely the intracellular content (to the exclusion of the membrane debris, sugars, fibers and fats) and the preservation, especially, of the quality of the protein load.

In the case of the microalga of the genus Tetraselmis sp, Anja Schwenzfeier et al (Bioresource Technology, 2011, 102, 9121-9127) proposed a method guaranteeing the solubility and the quality of the aminogram of the proteins isolated and with contaminants (such as coloring substances) removed, comprising the following steps:

-   -   cell disintegration by bead mill,     -   centrifugation of the milled microalgal suspension,     -   dialysis of the supernatant,     -   passage over ion-exchange resin,     -   dialysis of the eluate,     -   discoloration, then,     -   washing and resuspending.

However, this laboratory method (for treating 24 g of biomass) cannot be scaled up to an industrial scale, where the bead mill method is rather used to recover a complete biomass.

Moreover, this method is not suited to microalgae which contain, in their biomass, a not insignificant lipid content (for example in Chlorella protothecoides the lipid content is more than 15%).

Indeed, even after this “relatively soft” breaking of the cell wall, the milled cell material is in the form of a relatively stable complex “oil in water” emulsion.

Cell components are therefore rather conventionally extracted at this stage by solvent or mechanically, but to the detriment of their integrity.

A first solution proposed by the prior art, and moreover tested by the Applicant company, consists in coupling the mechanical milling with an evaporation, in order to attempt to destabilize the emulsion, then in separating the fatty fraction by centrifugation.

However, the poor quality of the separation step (basic creaming) makes this phase separation method quite inefficient.

Even though the addition of ethanol, recommended at this stage (20-30%/raw), improves the destabilization of the emulsion, it only however enables defatting of the order of 50%, even at low yield.

Moreover, the mechanical route is particularly difficult or even impossible to carry out when the lipid fraction is bound to the protein/polysaccharide matrix.

Another solution proposes using neutral solvents. However, it has heavy constraints (quality, safety, regulations, etc.).

Subject of the Invention

The result of this is that there is an unfulfilled need for a technology for extracting and stabilizing cell components of the microalgae of interest, said cell components being released by mechanical milling.

The Applicant company has found that this need could be fulfilled by proposing an alternative method to those known from the prior art, by combining a method for mechanically milling the microalgal cells with steps for destructuring the lipid fraction produced by a treatment chosen from the group of alkaline and enzymatic treatments, followed by a step of centrifugation.

The defatted soluble fraction is then clarified by microfiltration then ultrafiltered to obtain the protein isolate.

The present invention thus relates to a method for preparing a protein isolate from the biomass of microalgae of the Chlorella genus, comprising the following steps:

-   -   providing a microalgal biomass produced by fermentation,     -   washing the biomass so as to eliminate the interstitial soluble         compounds, and concentration,     -   mechanical milling of the washed and concentrated biomass,         carried out in a horizontal bead mill type system, to obtain an         emulsion,     -   destructuring the emulsion obtained in this way,     -   triphase separation so as to separate the soluble fraction from         the fractions containing lipids and cell debris,     -   recovery, and optional clarification, of the soluble fraction         obtained in this way, especially by microfiltration so as to         remove residual insoluble substances therefrom, so as to obtain         the soluble protein isolate,     -   optional ultrafiltration of the clarified soluble fraction on a         membrane with a cut-off threshold of less than 5 kDa, preferably         of between 1 and 5 kDa, so as to obtain a soluble protein         isolate,     -   optional neutralization at pH 7,     -   evaporation, pasteurization and atomization of said protein         isolate.

Choice of the Microalgal Biomass

Preferably, the microalgae of the Chlorella genus are chosen from the group consisting of Chlorella vulgaris, Chlorella sorokiniana and Chlorella protothecoides, and are more particularly Chlorella protothecoides.

In one particular embodiment, the strain is Chlorella protothecoides (strain UTEX 250—The Culture Collection of Algae at the University of Texas at Austin—USA).

In another particular embodiment, the strain is Chlorella sorokiniana (strain UTEX 1663—The Culture Collection of Algae at the University of Texas at Austin—USA).

The culturing under heterotrophic conditions and in the absence of light conventionally results in the production of a Chlorella biomass having a protein content (evaluated by measuring the nitrogen content N×6.25) of 45% to 70% by weight of dry cells.

As will be exemplified hereinafter, this culturing is carried out in two steps:

-   -   preculturing in a medium containing glucose and yeast extract         for 72 h at 28° C. with agitation, then     -   culturing for production of the biomass per se in glucose and         yeast extract for more than 36 h at 28° C., with agitation and         at pH 6.5 adjusted with aqueous ammonia, which results in         approximately 80 g/l of biomass with a protein content         (evaluated by N×6.25) of the order of 52% by weight of dry         cells.

The biomass is then collected by solid-liquid separation, by frontal or tangential filtration or by any means known, moreover, to those skilled in the art.

Advantageously, the Applicant company then recommends washing and concentrating the biomass so as to eliminate the interstitial soluble compounds by a succession of concentration (by centrifugation)/dilution of the biomass.

On the industrial scale, in-line dilution and separation by centrifugation in one or two stages is advantageously chosen.

For the purposes of the invention, the term “interstitial soluble compounds” is intended to mean all the soluble organic contaminants of the fermentation medium, for example the hydrosoluble compounds such as the salts, the residual glucose, the oligosaccharides with a degree of polymerization (or DP) of 2 or 3, or the peptides.

This biomass purified in this way of its interstitial soluble compounds is then preferentially adjusted to a dry matter of between 15% and 30% by weight, preferably to a dry matter of between 20% and 30%.

For the remainder of the method of the invention, the biomass obtained in this way may be used as is, or thermally permeabilized (by a high-temperature short-time or HTST method—also developed by the Applicant company and protected in one of its as yet unpublished applications) so as to release the content of soluble peptides therefrom.

The residual proteins of this biomass may be extracted by the subsequent following steps.

Biomass Milling

The Applicant company recommends using (horizontal) bead mill technology.

More particularly, the milling may advantageously be carried out according to a method which the Applicant company has developed and protected in one of its as yet unexamined applications, in which:

-   -   the zirconium silicate beads have an apparent density of between         2 and 3.5 kg/l, and     -   the filling rate of the milling chamber is greater than or equal         to 80%.

The milling is carried out in continuous mode, for example by successive passes in series.

The density of the microalgae to be milled is chosen at a level of less than 250 g/l.

At the end of milling, an emulsion is obtained.

Destructuring of the Emulsion and Separation of its Components

The separation of the components of the emulsion in order to extract the peptide or polypeptide fraction of interest therefrom requires destructuring/destabilization of the emulsion resulting from the cell milling (complex mixture of lipids, proteins—peptides and polypeptides—and cell debris).

This destructuring/destabilization of the emulsion may be facilitated:

-   -   either by enzymatic predigestion, especially by specific         proteases, by treatment with polar solvent and/or by controlled         alkaline treatment targeting the protein fraction of the         emulsion,     -   or by adjusting the pH and the temperature, by treatment with a         polar solvent and/or by enzymatic digestion, especially of         cellulase type, targeting the interface with the lipid fraction         of the emulsion.

Thus, the milled cell material is conditioned in a stirred reactor fitted with a low shear stirring module, so as to limit emulsification while enabling homogeneous mixing promoting the specific treatment chosen (setting a pH, action of the lytic enzyme, etc.).

For example, in the case of a treatment which aims to destabilize the emulsion by treating the protein fraction in mixture via the enzymatic route, for example by a basic protease, the temperature and the pH of the emulsion are adjusted to the reaction conditions for said protease:

-   -   the temperature is adjusted to a value of greater than 30° C.,         preferably of the order of 60° C., and     -   the pH is adjusted to a value of greater than 7, preferably of         the order of 8 (or even optionally of the order of 10 if only         the action of pH is being utilized).

The duration of the reaction is between 2 and 8 h.

At the end of the lysis, ethanol at more than 5% (v/v) may be added to the reaction mixture as destabilizing agent for the emulsion (in the case of an oil in water emulsion).

The emulsion destabilized in this way may be (partially) split up by triphase separation, for example by centrifugation.

Thus, 3 phases are obtained:

-   -   an upper lipid cream,     -   an aqueous/intermediate (=“raw” soluble substances) soluble         compounds (and residual insoluble substances) phase, and     -   a pellet concentrating the cell debris.

The soluble fraction is essentially composed of a predominant protein fraction, soluble sugars, salts and residual lipid globules.

Membrane Separation

To release peptides and polypeptides, the method of the invention next leads to the isolation of the proteins of interest, preferably by membrane fractionation.

The Applicant company thus recommends carrying out the process in three steps:

-   -   recovery and clarification of the soluble fraction obtained in         this way by microfiltration so as to remove residual insoluble         substances therefrom,     -   ultrafiltration of the clarified soluble fraction on a membrane         with a cut-off threshold of less than 5 kDa, preferably of         between 1 and 5 kDa, and     -   optional neutralization at a pH of between 6 and 8, preferably         at a value of 7.

Utilizing these pathways makes it possible to purify the soluble peptides and polypeptides of their residual salts and sugars.

Precipitation at the pI

Alternatively, to isolate the peptides and polypeptides of interest, the choice may be made to carry out the process in three steps:

-   -   precipitating the proteins at their pI, by adjusting the pH of         the medium to a value of between 4 and 5,     -   centrifugation or microfiltration in order to recover the         precipitated proteins, and     -   dissolving in water at a pH of between 6 and 8, preferably 7.

It should be noted that although the latter two steps make it possible, according to the method of the invention, to obtain protein isolates having a protein content of more than 80%, preferably of more than 90% by weight, they lead, by their implementational methods, to compositions distinct in nature.

Obtaining the Isolate in Powder Form

The protein isolate in soluble form obtained in this way may be:

-   -   concentrated by evaporation,     -   pasteurized, and finally     -   atomized.

The invention will be understood more clearly from the following examples which are intended to be illustrative and nonlimiting.

EXAMPLES Example 1: Production of Chlorella protothecoides by Fed-Batch Fermentation

The strain used is Chlorella protothecoides UTEX 250

Preculture:

-   -   500 ml of medium in a 2 l conical flask;     -   Composition of the medium (in g/l):

TABLE 1 Macro- Glucose 40 elements K₂HPO₄ 3 (g/l) Na₂HPO₄ 3 MgSO₄•7H₂O 0.25 (NH₄)₂SO₄ 1 Citric acid 1 Clerol FBA 3107 (antifoam) 0.1 Micro- CaCl₂•2H₂O 30 elements FeSO₄•7H₂O 1 and MnSO₄•1H₂O 8 Vitamins CoSO₄•7H₂O 0.1 (mg/l) CuSO₄•5H₂O 0.2 ZnSO₄•7H₂O 0.5 H₃BO₃ 0.1 Na₂MoO₄•2H₂O 0.4 Thiamine HCl 1 Biotin 0.015 B12 0.01 Calcium pantothenate 0.03 p-Aminobenzoic acid 0.06

Incubation is carried out under the following conditions: duration: 72 h; temperature: 28° C.; agitation: 110 rpm (Infors Multitron incubator).

The preculture is then transferred to a 30 l Sartorius type fermenter.

Culture for Biomass Production

The medium is as follows:

TABLE 2 Macro- Glucose 40 elements KH₂PO₄ 1.8 (g/l) NaH₂PO₄ 1.4 MgSO₄•7H₂O 3.4 (NH₄)₂SO₄ 0.2 Clerol FBA 3107 (antifoam) 0.3 Micro- CaCl₂•2H₂O 40 elements FeSO₄•7H₂O 12 and MnSO₄•1H₂O 40 Vitamins CoSO₄•7H₂O 0.1 (mg/l) CuSO₄•5H₂O 0.5 ZnSO₄•7H₂O 50 H₃BO₃ 15 Na₂MoO₄•2H₂O 2 Thiamine HCl 6 Biotin 0.1 B12 0.06 Calcium pantothenate 0.2 p-Aminobenzoic acid 0.2

The initial volume (Vi) of the fermenter is adjusted to 17 l after inoculation. It is brought to a final volume of approximately 20-25 l.

The parameters for performing the fermentation are as follows:

TABLE 3 Temperature 28° C. pH 5.0-5.2 by 28% w/w NH₃ pO₂ 20% ± 5% (maintained by shaking) Shaking Minimum 300 rpm Air flow rate 15 l/min

When the residual glucose concentration falls below 10 g/l, glucose in the form of a concentrated solution at approximately 800 g/l is introduced so as to maintain the glucose content between 0 and 20 g/l in the fermenter.

Results

In 40 h, 80 g/l of biomass containing 52% of proteins are obtained.

Example 2. Milling the Chlorella protothecoides Biomass and Recovery of the Soluble Fraction—Destructuring of the Emulsion by Treatment of the Peptide and Polypeptide Fraction

The biomass obtained according to example 1 is washed and concentrated by centrifugation so as to be brought to a dry matter content of 220 g/l and to a purity of more than 90% (purity defined by the ratio of the dry matter of the biomass to the total dry matter).

It is then milled by bead milling (horizontal bead mill) with zirconium silicate beads (0.6 mm diameter, apparent density 2.4).

The milled biomass is then agitated in a reactor fitted with a marine impeller and baffles. The temperature is adjusted to 60° C. and the pH to 8 with potassium hydroxide. A basic protease in combination with a cellulase are added, with these reaction conditions being maintained for a duration of 6 h.

The emulsion is then centrifuged on a triphase centrifuge which makes it possible to obtain 3 phases: an upper lipid cream, an aqueous/intermediate (=“raw” soluble substances) soluble compounds (and residual insoluble substances) phase, and a pellet concentrating the cell debris.

The fraction of raw soluble substances is clarified by microfiltration. The microfiltration permeate “P1” has a titer between 55% and 70% of peptides and proteins (expressed as total amino acids) and is then ultrafiltered on a membrane with a <5 kDa cut-off threshold.

The ultrafiltration retentate “R2” obtained in this way contains more than 80% of peptides having a molecular weight of greater than or equal to 5 kDa.

The permeate “P2” contains peptides having a molecular weight of less than 5 kDa and oligosaccharides and residual salts.

This permeate “P2” can then especially be filtered on a reverse osmosis membrane (having a degree of NaCl rejection of 93%), so as to obtain:

-   -   a retentate “R3”, containing peptides having a molecular weight         of less than 5 kDa and oligosaccharides of DP 2, such as         sucrose; and     -   a permeate “R3”, containing oligosaccharides of DP 1, salts,         free amino acids and organic acids.

The protein isolate “R2” is then:

-   -   neutralized to pH 7 with potassium hydroxide,     -   concentrated by evaporation to 35% dry matter (DM),     -   pasteurized, then     -   atomized.

Example 3. Milling the Chlorella protothecoides Biomass and Recovery of the Soluble Fraction—Destructuring of the Emulsion by Treatment of the Lipid Fraction

According to the same sequence as in example 2, the milled biomass is agitated in a reactor fitted with a marine impeller and baffles. The temperature is adjusted to 50° C. without adjusting the pH (naturally between 5 and 6).

A cellulase having optimum activity in this pH and temperature range is added, with these reaction conditions being maintained for a duration of 6 h.

At the end of the reaction, the pH is adjusted to 8 before the separation into 3 phases.

The remainder of the operations is described in example 2. 

The invention claimed is:
 1. A method for preparing a protein isolate from the biomass of microalgae of the Chlorella genus, comprising: providing a microalgal biomass produced by fermentation of microalgae of the Chlorella genus, wherein the fermentation is carried out for more than 36 hours at 28° C. with agitation and at a pH of 6.5, washing the microalgal biomass so as to eliminate interstitial soluble compounds and concentrate the microalgal biomass, mechanical milling of the washed and concentrated microalgal biomass, carried out in a horizontal bead mill type system, to obtain an emulsion, destructuring the obtained emulsion by (i) or (ii), thus resulting in a destructured emulsion: (i) by enzymatic predigestion, by treatment with polar solvent, and/or by controlled alkaline treatment targeting the protein fraction of the emulsion for a duration of 2-8 hours at a temperature of more than 30° C. and a pH of greater than 7; or (ii) by treatment with a polar solvent, and/or by enzymatic digestion targeting the interface with the lipid fraction of the emulsion for a duration of 6 hours at a temperature of 50° C. and a pH of 5-6; triphase separation so as to separate the soluble fraction from the fractions containing lipids and cell debris, recovering the soluble fraction obtained in this way in order to obtain the soluble protein isolate, then evaporation, pasteurization and atomization of the protein isolate.
 2. The method as claimed in claim 1, wherein the microalgae of the Chlorella genus are selected from the group consisting of Chlorella vulgaris, Chlorella sorokiniana, and Chlorella protothecoides.
 3. The method as claimed in claim 1, wherein the triphase separation of the destructured emulsion is carried out by centrifugation.
 4. The method as claimed in claim 1, wherein the soluble protein isolate is obtained from the soluble fraction by: clarifying the soluble fraction by microfiltration so as to remove residual insoluble substances therefrom, ultrafiltration of the clarified soluble fraction on a membrane with a cut-off threshold of less than 5 kDa, optionally, neutralizing at a pH of between 6 and
 8. 5. The method as claimed in claim 1, wherein the soluble protein isolate is obtained from the soluble fraction by: precipitating the proteins at their pI, by adjusting the pH of the medium to a value of between 4 and 5, centrifugation or microfiltration in order to recover the precipitated proteins, and dissolving in water at a pH of between 6 and
 8. 6. The method as claimed in claim 2, wherein the microalgae of the Chlorella genus is Chlorella protothecoides.
 7. The method as claimed in claim 2, wherein the triphase separation of the destructured emulsion is carried out by centrifugation.
 8. The method as claimed in claim 2, wherein the soluble protein isolate is obtained from the soluble fraction by: clarifying the soluble fraction by microfiltration so as to remove residual insoluble substances therefrom, ultrafiltration of the clarified soluble fraction on a membrane with a cut-off threshold of less than 5 kDa, and optionally, neutralizing at a pH of between 6 and
 8. 9. The method as claimed in claim 2, wherein the soluble protein isolate is obtained from the soluble fraction by: precipitating the proteins at their pI, by adjusting the pH of the medium to a value of between 4 and 5, centrifugation or microfiltration in order to recover the precipitated proteins, and dissolving in water at a pH of between 6 and
 8. 10. The method as claimed in claim 5, wherein the dissolving in water is carried out at a pH of
 7. 